Anti-cancer antibodies with reduced complement fixation

ABSTRACT

The invention provides modified antibodies directed against GD2 that have diminished complement fixation relative to antibody-dependent, cell-mediated cytotoxicity, which is maintained. The modified antibodies of the invention may be used in the treatment of tumors such as neuroblastoma, glioblastoma, melanoma, small-cell lung carcinoma, B-cell lymphoma, renal carcinoma, retinoblastoma, and other cancers of neuroectodermal origin.

RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 60/538,348, filed Jan. 22, 2004, the entire disclosureof which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to the field of anti-cancer antibodiesfor targeting tumor cells. More specifically, the invention relates toanti-GD2 antibodies for targeting tumor cells expressing the glycolipidGD2.

BACKGROUND OF THE INVENTION

A common method of treating cancer involves using antibodies to attacktumor cells by specifically targeting tumor cell associated antigens.One specific example of this method involves using anti-GD2 antibodiestargeted against GD2, a glycolipid which is highly expressed in certaintumor cells, such as glioblastoma, melanoma, small-cell lung carcinoma,and neuroblastoma. Specifically, anti-GD2 antibodies, such as 14.18,have been tested against neuroblastoma and osteosarcoma tumors (Yu etal., J. Clin. Oncol., [1998]; 16: 2169-80), with encouraging results.However, because GD2 is also expressed in nerve endings, pain is aserious side effect of anti-GD2 antibody treatment (Kushner et al., J.Clin. Oncol., [2001]; 19: 4189-94; Frost et al., Cancer, [1997]; 80:317-33; Yu et al., J. Clin. Oncol., [1998]; 16: 2169-80). Thus, there isa need in the art for antibodies directed against GD2 that exhibitreduced side effects, while maintaining effectiveness in treatingcancers that express the GD2 glycolipid.

SUMMARY OF THE INVENTION

The invention relates to proteins comprising antibody moieties in whichthe proteins bind to the GD2 glycolipid and induce antibody-dependentcell-mediated cytotoxicity (ADCC), but have reduced complement fixation.When administered to patients, the antibodies and related proteins ofthe invention generally result in patients experiencing lower painlevels when compared to pain levels generated by administration of thecorresponding proteins not modified in accordance with the invention. Asa result, in some treatment modalities, patient suffering is alleviatedand quality of life is improved. In other treatment modalities, the doseof the therapeutic protein of the invention is higher than thecorresponding antibody-based protein without the modifications of theinvention.

In one embodiment of the invention, antibody-based proteins comprisingan Fc region and a variable region capable of binding GD2 are used. In afurther embodiment the Fc region is derived from IgG, more specificallyIgG1. In further embodiments, the antibody-based proteins of theinvention may include CH1 domains and/or CL domains. However, thepresence of CH1 domains or CL domains is optional and not necessary. Ina further embodiment, the variable region of the antibody based proteinis connected to an Fc region by a linker, more specifically apolypeptide linker. The polypeptide linker may comprise glycine and/orserine. In one embodiment, the linker has the polypeptide sequenceGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:10). In a further embodiment, thevariable region is at least 60%, 70%, 80%, or 90% identical to thevariable region of the canonical 14.18 antibody (Yu et al., J. Clin.Oncol., [1998]; 16: 2169-80; U.S. Patent Application Publication No.2003-0157054-A1).

In another class of embodiments, modifications to the antibody-basedproteins of the invention that enhance antibody-dependent cell-mediatedcytotoxicity (ADCC) relative to complement fixation may also be used. Ina preferred embodiment, the Fc region has a modification that reduces orabolishes complement fixation, e.g., relative to levels of ADCC. Inanother embodiment, the Fc region of IgG1 has been modified by themutation Lys322Ala. Other mutations that reduce complement fixation maybe used, and the mutations may be amino acid substitutions as well asdeletions or insertions of amino acids. In a further embodiment, theinvention provides proteins with enhanced levels of bisected N-linkedoligosaccharide in the Fc moiety of an anti-GD2-based protein. In afurther embodiment, the invention also provides protein productionmethods that enhance the formation of bisected N-linked oligosaccharidesin the Fc moiety of an anti-GD2-based protein. In a particularembodiment, anti-GD2 antibodies are expressed in the rat-derived cellline YB2/0, which results in antibodies having higher ADCC activity thananti-GD2 antibodies expressed from most other cell lines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the wild-type amino acid sequence of the human 14.18 IgG1mature heavy chain (SEQ ID NO:1).

FIG. 2 depicts an amino acid sequence of the human 14.18 IgG1 matureheavy chain with a K322A mutation (SEQ ID NO:5). The substituted alanineresidue at 322 is underlined.

FIG. 3 depicts the amino acid sequence of the mature human 14.18 IgG1light chain. (SEQ ID NO:2)

FIG. 4 depicts a nucleic acid sequence (SEQ ID NO:3), with introns,encoding a human 14.18 IgG1 mature heavy chain.

FIG. 5 depicts a nucleic acid sequence (SEQ ID NO:4), with introns,encoding a human 14.18 IgG1 mature light chain.

FIG. 6 depicts the results of an antibody-dependent cellularcytotoxicity assay performed on GD2 expressing M-21 cells. The x-axisindicates the concentration of antibody in ng/ml while the y-axisindicates the percent lysis of target cells.

FIG. 7 depicts the results of an antibody-dependent cellularcytotoxicity assay performed on GD2 expressing LN-229 cells. The x-axisindicates the concentration of antibody in ng/ml while the y-axisindicates the percent lysis of target cells.

FIG. 8 depicts the results of an antibody-dependent cellularcytotoxicity assay performed on non-GD2 expressing EpCAM+A431 cells. Thex-axis indicates the concentration of antibody in ng/ml while the y-axisindicates the percent lysis of target cells.

FIG. 9 depicts the results of a complement dependent cytotoxicity assayperformed on GD2 expressing M-21 cells. The x-axis indicates theconcentration of antibody in ng/ml while the y-axis indicates thepercent lysis of target cells.

FIG. 10 depicts the results of a complement-dependent cytotoxicity assayperformed on GD2 expressing LN-229 cells. The x-axis indicates theconcentration of antibody in ng/ml while the y-axis indicates thepercent lysis of target cells.

FIG. 11 depicts the amino acid sequence of the mature fusion proteinhuman 14.18 sFv(VL-VH)-Fc (SEQ ID NO:9) which is an sFv antigen bindingportion connected via a polypeptide linker with the amino acid sequenceGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:10) to an Fc fragment consisting ofhinge, CH2 and CH3 domains of IgG1.

FIG. 12 depicts a nucleic acid sequence encoding the mature human 14.18sFv(VL-VH)-Fc antibody construct (SEQ ID NO:8) of FIG. 10.

DETAILED DESCRIPTION OF THE INVENTION

The GD2 glycolipid is expressed on a variety of tumor types, but isessentially not expressed in normal tissues, with the exception of someexpression at nerve endings. Antibodies directed against the GD2glycolipid antigen have been tested in cancer patients with somesuccess. However, presumably because of the expression of GD2 inneurons, pain is a major side effect of anti-GD2 antibody treatment, andis consequently a dose-limiting toxicity. The present invention providesanti-GD2 antibodies and related molecules that induce less pain.

As used herein, the term glycolipid GD2 or GD2 antigen is defined as aglycolipid capable of specific binding to an anti-GD2 antibody asdefined herein. The term anti-GD2 antibody is defined as an antibodycapable of specific binding to the antigen glycolipid GD2. As usedherein, the terms “bind specifically,” specifically bind,” and “specificbinding” are understood to mean that the antibody has a binding affinityfor a particular antigen of at least about 10⁶ M⁻¹, more preferably, atleast about 10⁷ M⁻¹, more preferably at least about 10⁸ M⁻¹, and mostpreferably at least about 10¹⁰ M⁻¹.

As used herein, the terms “antibody” is understood to mean (i) an intactantibody (for example, a monoclonal antibody or polyclonal antibody),(ii) antigen binding portions thereof, including, for example, an Fabfragment, an Fab′ fragment, an (Fab′)₂ fragment, an Fv fragment, asingle chain antibody binding site, an sFv, (iii) bi-specific antibodiesand antigen binding portions thereof, and (iv) multi-specific antibodiesand antigen binding portions thereof. Furthermore, the term “antibody”encompasses any of an Fab fragment, an Fab′ fragment, an (Fab′)₂fragment, an Fv fragment, a single chain antibody binding site, or ansFv fragment linked to an Fc fragment or any portion of an Fc fragment.The linkage can be accomplished through use of linker peptide sequencesknown in the art. An antibody of the invention may be naturallyoccurring or synthetic, such as a recombinant antibody.

As used herein, the term “immunoglobulin” is understood to mean anaturally occurring or synthetically produced polypeptide homologous toan intact antibody (for example, a monoclonal antibody or polyclonalantibody) or fragment or portion thereof, such as an antigen-bindingportion thereof. Immunoglobulin according to the invention may be fromany class such as IgA, IgD, IgG, IgE, or IgM. IgG immunoglobulins can beof any subclass such as IgG1, IgG2, IgG3, or IgG4. The termimmunoglobulin also encompasses polypeptides and fragments thereofderived from immunoglobulins. Immunoglobulins can be naturally occurringor synthetically produced, such as recombinant immunoglobulins.

The constant region of an immunoglobulin is defined as anaturally-occurring or synthetically-produced polypeptide homologous tothe immunoglobulin C-terminal region, and can include a CH1 domain, ahinge, a CH2 domain, a CH3 domain, or a CH4 domain, separately or in anycombination. As used herein, “Fc portion” encompasses domains derivedfrom the constant region of an anti-GD2 antibody, including a fragment,analog, variant, mutant or derivative of the constant region. Suitableimmunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes. Theconstant region of an immunoglobulin is defined as a naturally-occurringor synthetically-produced polypeptide homologous to the immunoglobulinC-terminal region, and can include a CH1 domain, a hinge, a CH2 domain,a CH3 domain, or a CH4 domain, separately or in any combination. In thepresent invention, the Fc portion typically includes at least a CH2domain. For example, the Fc portion can include hinge-CH2-CH3.Alternatively, the Fc portion can include all or a portion of the hingeregion, the CH2 domain and/or the CH3 domain and/or CH4 domain.

The term variable fragment or Fv as used herein is defined as anaturally occurring or synthetically produced polypeptide homologous tothe heavy chain variable region and/or the light chain variable region.More specifically, an Fv can be an sFv or single chain variable fragmentwherein the heavy chain variable region and the light chain variableregion are linked together by a polypeptide moiety. Such polypeptidelinker sequences are known in the art.

Anti-tumor activity of antibodies generally occurs via either complementdependent cytotoxicity (CDC or complement fixation) or through anti-bodydependent cell-mediated cytotoxicity (ADCC). These two activities areknown in the art as “effector functions” and are mediated by antibodies,particularly of the IgG class. All of the IgG subclasses (IgG1, IgG2,IgG3, IgG4) mediate ADCC and complement fixation to some extent, withIgG1 and IgG3 being most potent for both activities (Chapter 3, Table 3in Paul, Essential Immunology 4^(th) Ed., p. 62). ADCC is believed tooccur when Fc receptors on natural killer (NK) cells bind to the Fcregion of antibodies bound to antigen on a cell's surface. Fc receptorbinding signals the NK cell to kill the target cell. CDC is believed tooccur by multiple mechanisms; one mechanism is initiated when anantibody binds to an antigen on a cell's surface. Once theantigen-antibody complex is formed, the C1q molecule is believed to bindthe antigen-antibody complex. C1q then cleaves itself to initiate acascade of enzymatic activation and cleavage of other complementproteins which then bind the target cell surface and facilitate itsdeath through, for example, cell lysis and/or ingestion by a macrophage.

A key insight of the invention is that CDC causes the side effect ofpain. Without wishing to be bound by theory, neurons may be particularlysensitive to complement fixation because this process involves thecreation of channels in a cell membrane, allowing an uncontrolled ionflux. In pain-sensing neurons, even a small amount of complementfixation may be significant to generate action potentials. Thus, anyamount of CDC resulting from anti-GD2 antibody binding on neurons willresult in pain. According to the invention, it is advantageous to reducecomplement fixation so as to reduce the level of side effects in apatient.

However, if one reduces or eliminates CDC, effective anti-tumor activityof the anti-GD2 antibody requires that levels of ADCC be maintained oreven increased. A second key finding of the invention is that theantitumor activity of anti-GD2 antibodies results primarily from ADCC,and not substantially from complement fixation. Therefore, a key aspectof the invention is that it is possible to reduce or eliminate CDCfunction of an anti-GD2 antibody without eliminating the anti-tumorcapabilities of the anti-GD2 antibody. In other words, an anti-GD2antibody modified to reduce or eliminate complement fixation will stillhave anti-tumor capabilities and therefore can be effective at treatingtumor growth. Consequently, the invention provides mutations in anti-GD2antibodies that reduce complement fixation to a great extent whilehaving a minimal effect on ADCC, such as mutation of lysine 322 toalanine (K322A) or another amino acid (Thommesen et al., Mol. Immunol.,[2000]; 37(16): 995-1004).

The anti-GD2 antibodies of the invention can be produced usingrecombinant expression vectors known in the art. The term “expressionvector” refers to a replicable DNA construct used to express DNAencoding the desired anti-GD2 antibody and including a transcriptionalunit of (1) genetic element(s) having a regulatory role in geneexpression, for example, promoters, operators, or enhancers, operativelylinked to (2) a DNA sequence encoding the desired anti-GD2 antibodywhich is transcribed into mRNA and translated into protein, and (3)appropriate transcription and translation initiation and terminationsequences. The choice of promoter and other regulatory elementsgenerally varies according to the intended host cell.

In a preferred example, the nucleic acid encoding the modified anti-GD2antibody is transfected into a host cell using recombinant DNAtechniques. In the context of the present invention, the foreign DNAincludes a sequence encoding the inventive proteins. Suitable host cellsinclude prokaryotic, yeast or higher eukaryotic cells.

The recombinant anti-GD2 antibodies can be expressed in yeast hosts,preferably from Saccharomyces species, such as S. cerevisiae. Yeasts ofother genera such as Pichia or Kluyveromyces may also be employed. Yeastvectors will generally contain an origin of replication from a yeastplasmid or an autonomously replicating sequence (ARS), a promoter, DNAencoding the anti-GD2 antibody, as well as sequences forpolyadenylation, transcription termination, and a selection gene.Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-4-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase and glucokinase.

Various mammalian or insect cell culture systems can be employed toexpress the recombinant protein of the invention. Baculovirus systemsfor production of proteins in insect cells are well known in the art.Examples of suitable mammalian host cell lines include NS/0 cells, Lcells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines.Additional suitable mammalian host cells include CV-1 cells (ATCC CCL70)and COS-7 cells, both derived from monkey kidney. Another suitablemonkey kidney cell line, CV-1/EBNA, was derived by transfection of theCV-1 cell line with a gene encoding Epstein-Barr virus nuclear antigen-1(EBNA-1) and with a vector containing CMV regulatory sequences (McMahanet al., EMBO J., [1991]; 10: 2821-32). The EBNA-1 gene allows forepisomal replication of expression vectors, such as HAV-EO or pDC406,that contain the EBV origin of replication.

According to this invention, a particularly useful cell line forexpression of anti-GD2 antibodies is the YB2/0 cell line (Shinkawa etal., J. Biol. Chem., [2003]; 278: 3466-3473). Antibodies produced fromthis cell line have enhanced ADCC. When produced from this cell line,antibodies of the invention have a different N-linked oligosaccharidethan the oligosaccharide seen in antibodies produced from other celllines described above. Particular embodiments of the invention includeanti-GD2 antibodies with non-mutant constant regions produced in YB2/0,as well as anti-GD2 antibodies with constant regions bearing mutationsthat reduce complement fixation, such as Lys322Ala, also produced inYB2/0 cells.

Mammalian expression vectors can include non-transcribed elements suchas an origin of replication, a suitable promoter and enhancer linked tothe gene to be expressed, and other 5′ or 3′ flanking non-transcribedsequences, and 5′ or 3′ non-translated sequences, such as necessaryribosome binding sites, a poly-adenylation site, splice donor andacceptor sites, and transcriptional termination sequences. Commonly usedpromoters and enhancers are derived from Polyoma, Adenovirus 2, SimianVirus 40 (SV40), and human cytomegalovirus. DNA sequences derived fromthe SV40 viral genome, for example, SV40 origin, early and latepromoter, enhancer, splice, and polyadenylation sites may be used toprovide the other genetic elements required for expression of aheterologous DNA sequence.

When secretion of the modified antibody from the host cell is desired,the expression vector can include DNA encoding signal or leaderpeptides, preferably placed N-terminally to both heavy and light chains.In the present invention the native signal sequences of the antibody Vregions can be used, or alternatively, a heterologous signal sequencemay be added, such as the signal sequence from interleukin-4.

The present invention also provides a process for preparing therecombinant proteins of the present invention including culturing a hostcell transformed with an expression vector comprising a DNA sequencethat encodes the anti-GD2 antibody under conditions that promoteexpression. The desired protein is then purified from culture media orcell extracts. For example, supernatants from expression systems thatsecrete recombinant protein into the culture medium can be firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a suitable purification matrix, as known in the art.

An “isolated” or “purified” modified anti-GD2 antibody or biologicallyactive portion thereof is substantially free of cellular material orother contaminating proteins from the cell or tissue source from whichthe modified anti-GD2 antibody is derived, or substantially free fromchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof modified anti-GD2 antibody in which the protein is separated fromcellular components of the cells from which it is isolated orrecombinantly produced. In one embodiment, the language “substantiallyfree of cellular material” includes preparations of modified anti-GD2antibody having less than about 30% (by dry weight) of non-antibody(also referred to herein as a “contaminating protein”), more preferablyless than about 20% of non-antibody protein, still more preferably lessthan about 10% of non-antibody protein, and most preferably less thanabout 5% non-antibody protein. When the modified anti-GD2 antibody orbiologically active portion thereof is purified from a recombinantsource, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation.

The term “substantially pure modified anti-GD2 antibody” refers to apreparation in which the modified anti-GD2 antibody constitutes at least60%, 70%, 80%, 90%, 95% or 99% of the proteins in the preparation.

Methods of Treatment using Anti-GD2 Antibody Proteins

The modified anti-GD2 antibodies of the invention are useful in treatingcancers, such as GD2-expressing cancers. Such cancers include, but arenot limited to, neuroblastoma, glioblastoma, melanoma, small-cell lungcarcinoma, B-cell lymphoma, renal carcinoma, retinoblastoma, and othercancers of neuroectodermal origin.

Administration

The modified anti-GD2 antibodies of the invention can be incorporatedinto a pharmaceutical composition suitable for administration. Suchcompositions typically comprise the modified anti-GD2 antibodies and apharmaceutically-acceptable carrier. As used herein the language“pharmaceutically-acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Medicaments that contain the modified anti-GD2 antibodies of theinvention can have a concentration of 0.01 to 100% (w/w), though theamount varies according to the dosage form of the medicaments.

Administration dose depends on the body weight of the patients, theseriousness of the disease, and the doctor's opinion. However, it isgenerally advisable to administer about 0.01 to about 10 mg/kg bodyweight a day, preferably about 0.02 to about 2 mg/kg/day in case ofinjection, and more preferably about 0.5 mg/kg/day. The dose can beadministered once or several times daily according to the seriousness ofthe disease and the doctor's opinion.

Compositions of the invention are useful when co-administered with oneor more other therapeutic agents, for example, chemotherapeutic agentsthat are standard treatment in cancer therapy.

EXAMPLES Example 1 Expression of an Anti-GD2 Antibody with a Mutationthat Reduced Complement Fixation

An expression plasmid that expresses the heavy and light chains of thehuman 14.18 anti-GD2 antibody with reduced complement fixation due tomutation was constructed as follows. The expression plasmid for the14.18 anti-GD2 antibody was pdHL7-hu14.18. pdHL7 was derived from pdHL2(Gillies et al., J. Immunol. Methods, [1989]; 125: 191-202), and usesthe cytomegalovirus enhancer-promoter for the transcription of both theimmunoglobulin light and heavy chain genes. The K322A mutation in theCH2 region was introduced by overlapping Polymerase Chain Reactions(PCR) using pdHL7-hu14.18 plasmid DNA as template. The sequence of theforward primer was 5′-TAC AAG TGC GCT GTC TCC AAC (SEQ ID NO:6), wherethe underlined GCT encodes the K322A substitution, and the sequence ofthe reverse primer was 5′-T GTT GGA GAC AGC GCA CTT GTA (SEQ ID NO:7),where the underlined AGC is the anticodon of the introduced alanineresidue. The PCR product was cloned and, after sequence confirmation,the DNA containing the K322A mutation was excised as a 190 base-pair(bp) Sac II-Nae I restriction fragment (the restriction sites Sac II andNae I are located about 90 bp upstream and 100 bp downstream,respectively, of the K322A mutation), which was then used to replace thecorresponding fragment containing the K322 wild-type in thepdHL7-hu14.18 to give pdHL7-hu14.18(K322A). The expression vector forthe 14.18(K322A) antibody, pdHL7-hu14.18(K322A), was constructed in amanner analogous to the construction of pdHL7-hu14.18. However, oneskilled in the art may choose from a number of acceptable vectors toexpress hu14.18 K322A.

Example 2 Transfection and Expression of the Anti-GD2 Antibody

Electroporation was used to introduce the DNA encoding the anti-GD2antibody described above into a mouse myeloma NS/0 cell line or theYB2/0 cell line. To perform electroporation, cells were grown inDulbecco's modified Eagle's medium supplemented with 10%heat-inactivated fetal bovine serum, 2 mM glutamine andpenicillin/streptomycin. About 5×10⁶ cells were washed once with PBS andresuspended in 0.5 ml PBS. 10 μg of linearized plasmid DNA encoding themodified anti-GD2 antibody of Example 1 was then incubated with thecells in a Gene Pulser Cuvette (0.4 cm electrode gap, BioRad) on ice for10 min. Electroporation was performed using a Gene Pulser (BioRad,Hercules, Calif.) with settings at 0.25 V and 500 μF. Cells were allowedto recover for 10 min on ice, after which they were resuspended ingrowth medium and plated onto two 96 well plates.

Stably transfected clones were selected by their growth in the presenceof 100 nM methotrexate (MTX), which was added to the growth medium twodays post-transfection. The cells were fed two to three more times onevery third day, and MTX-resistant clones appeared in 2 to 3 weeks.Supernatants from clones were assayed by anti-Fc ELISA to identifyclones that produced high amounts of the anti-GD2 antibody. Highproducing clones were isolated and propagated in growth mediumcontaining 100 nM MTX. Typically, a serum-free growth medium, such asH-SFM or CD medium (Life Technologies), was used.

Example 3 Biochemical Analysis of the Anti-GD2 Antibody

Routine SDS-PAGE characterization was used to assess the integrity ofthe modified antibodies. The modified anti-GD2 antibodies were capturedon Protein A Sepharose beads (Repligen, Needham, Mass.) from the tissueculture medium into which they were secreted, and were eluted by boilingin protein sample buffer, with or without a reducing agent such asβ-mercaptoethanol. The samples were separated by SDS-PAGE and theprotein bands were visualized by Coomassie staining. Results fromSDS-PAGE showed that the modified anti-GD2 antibody proteins analyzedwere present substantially as a single band, indicating that there wasnot any significant amount of degradation.

Example 4 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) andComplement-Dependent Cytotoxicity (CDC) of Antibodies of the Invention

To demonstrate that the modified antibodies of the invention had thedesired properties, the ability of the antibodies to mediate ADCC wasexamined using standard procedures, essentially as described by Idusogieet al. (J. Immunol., [2000]; 164: 4178-4184). Antibodies were testedagainst two GD2-positive, EpCAM-negative cell lines (M-21 and LN-229)and, as a control, one GD2-negative, EpCAM-positive cell line (A431)using human PBMCs as effector cells in a standard chromium releaseassay. All antibodies had a human IgG1 isotype.

Human IgG2 versions of 14.18 anti-GD2 antibody expressed in NS/0 cells;14.18 with the K322A mutation, expressed in NS/0 cells; 14.18 with theK322A mutation, expressed in YB2/0 cells; and 14.18 configured as asingle-chain Fv fused to an Fc, expressed in NS/0 cells; were assayed todetermine their ADCC activity by measuring the percent of target M-21cells lysed according to standard methods described previously. TheKS-1/4 antibody, which does not bind target cells, was also assayed toserve as a control. As shown in FIG. 6, the K322A variant grown in YB2/0cells had the highest overall levels of ADCC as compared to all otherantibody constructs tested.

The same constructs were also tested in a similar assay using LN-229 GD2expressing cells as the target cells. The KS-1/4 antibody was used as acontrol. As shown in FIG. 7, the K322A variant grown in YB2/0 cells hadthe highest overall levels of ADCC as compared to all other antibodyconstructs tested.

As a control, the ADCC activity of the same anti-GD2 antibodies wastested against A431 cells which do not express the glycolipid GD2. Aswould be expected, the anti-GD2 antibodies showed little, if noactivity, whereas the KS-1/4 antibody which is known to have ADCCactivity against EpCAM expressing cells demonstrated increasing activityas concentrations of the antibody increased. (See FIG. 8). Thisindicates that the levels of lysis achieved in the assay of anti-GD2antibody with M-21 cells in the previous assay need not be adjusted forany background levels of lysis activity.

In order to test the ability of anti-GD2 antibodies ability to mediatecomplement dependent cytotoxicity (CDC), human IgG1 versions of 14.18expressed in NS/0 cells; two samples of 14.18 with the K322A mutation,expressed in NS/0 cells; 14.18 with the K322A mutation, expressed inYB2/0 cells; and 14.18 configured as a single-chain Fv fused to an Fc,expressed in NS/0 cells. Anti-GD2 antibodies of the invention wereexamined in M-21 and LN-229 cell lysis assays according to standardprocedures, essentially as described by Idusogie et al. (J. Immunol.,[2000]; 164: 4178-4184). A 1:10 dilution of human complement was used.The KS-1/4 antibody, which does not bind to the target cells, was againused as a control.

It was found that complement fixation mediated by the antibodies of theinvention was profoundly reduced. As shown in FIG. 9, only 14.18 grownin NS/0 cells had levels of complement fixation at low concentrations,whereas those antibodies containing the K322A mutation exhibited littleor no CDC activity at low concentrations. As shown in FIG. 10, only14.18 anti-GD2 antibodies grown in NS/0 cells demonstrated complementfixation activity against LN229 cells, as measured by the percent oftarget cells lysed. The K322A variants all demonstrated little or no CDCactivity at any concentration.

Taken together, the results of the ADCC and CDC assays indicate thatcertain modified anti-GD2 antibodies of the invention mediate ADCC, buthave significantly reduced levels of complement fixation, especially ascompared to typical anti-GD2 antibodies that have been used in humanclinical trials.

Example 5 Treatment of Mice Bearing GD2-Expressing Tumors with ModifiedAnti-GD2 Antibodies

To demonstrate the efficacy of the modified anti-GD2 antibodies of theinvention, the modified antibody of Example 1 is tested in a mouse modelof melanoma or neuroblastoma. Hu/SCID beige mice are used. The SCID andbeige mutations suppress the normal mouse immune system, so that humanimmune cells can be added to reconstitute the immune system. Humanperipheral blood mononucleocytes (PBMCs) are used. It is necessary touse human immune cells because the Fc region of human IgG1 is notrecognized by murine Fc receptors to achieve ADCC.

Cells expressing GD2 are then implanted into the mice. For example,cells are implanted subcutaneously, and their growth is monitored twiceper week with calipers to estimate tumor volume. As a model ofneuroblastoma, the GD2-expressing cell line NXS2 is used (Greene et al.,Proc. Natl. Acad. Sci. USA, [1975]; 72: 4923-27). As a model ofmelanoma, the cell line B16, modified to express GD2 is used (Haraguchiet al., Proc. Natl. Acad. Sci. USA, [1994]; 91: 10455-59).

Subcutaneous tumors are allowed to grow to a size of about 25 to 200cubic millimeters, and treatment is initiated. Because the serumhalf-life of the modified antibodies of the invention is several days,animals are treated only one to three times per week. It is found thatthe volumes of tumors in mice treated with either vehicle or a controlantibody increase rapidly, while the volumes of mice treated with themodified antibodies of the invention increase more slowly, or arestabilized, or in some cases shrink.

Example 6 Determination of the Maximum Tolerated Dose in a Phase IClinical Trial

To determine the maximum tolerated dose of a modified anti-GD2 antibodyof the invention, a Phase I clinical trial is performed essentially asdescribed in Yu et al. (J. Clin. Oncol., [1998]; 16: 2169-80). Themaximum tolerated dose of the human IgG1-based chimeric 14.18 antibodyreported by Yu et al. was found to be about 20 mg/m². The maximumtolerated dose of the modified anti-GD2 antibody of the invention isfound to be higher than 20 mg/m².

1. An isolated antibody which binds to GD2, the antibody comprising apolypeptide comprising the amino acid sequence of SEQ ID NO:5.
 2. Anisolated polypeptide which binds to GD2, the polypeptide comprising theamino acid sequence of SEQ ID NO:9.
 3. An isolated antibody which bindsto GD2, the antibody comprising the amino acid sequence of SEQ ID NO:5and the amino acid sequence of SEQ ID NO:2.